IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis
Robert A. Campbell, … , Anandi Krishnan, Matthew T. Rondina
Robert A. Campbell, … , Anandi Krishnan, Matthew T. Rondina
Published October 4, 2022
Citation Information: J Clin Invest. 2022;132(23):e153014. https://doi.org/10.1172/JCI153014.
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Research Article Hematology Inflammation

IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis

Abstract

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm–/– mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm–/– mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

Authors

Robert A. Campbell, Bhanu Kanth Manne, Meenakshi Banerjee, Elizabeth A. Middleton, Abigail Ajanel, Hansjorg Schwertz, Frederik Denorme, Chris Stubben, Emilie Montenont, Samantha Saperstein, Lauren Page, Neal D. Tolley, Diana L. Lim, Samuel M. Brown, Colin K. Grissom, Douglas W. Sborov, Anandi Krishnan, Matthew T. Rondina

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Figure 5

IFITM3 regulates fibrinogen endocytosis in megakaryocytes.

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IFITM3 regulates fibrinogen endocytosis in megakaryocytes. MEG-01s were ...
MEG-01s were left alone (no pZIP) or transfected with an empty vector control (pZIP+EV) or a vector containing IFITM3 (pZIP-IFITM3). (A) IFITM3 expression was determined (representative immunoblot shown from n = 3 independent experiments). (B and C) pZIP EV control or pZIP IFITM3-overexpressing MEG-01s were stimulated with PMA (100 nM, to induce differentiation) for 24 hours before incubation with 10 μg labeled fibrinogen. Fibrinogen endocytosis (percentage and MFI) was measured in mature CD41+ MEG-01s by flow cytometry (n = 4). Paired t test. (D and E) pZIP EV control or pZIP IFITM3–overexpressing MEG-01s were stimulated with PMA for 24 hours before incubation with labeled transferrin. Transferrin endocytosis (percentage and MFI) was measured in CD41+ MEG-01s by flow cytometry (n = 6). Paired t test. (F) MEG-01s were transfected with control shRNA (sh-NC) or a vector containing shRNA against IFITM3 (sh-IFITM3). sh-NC and sh-IFITM3 MEG-01s were treated with IFN-α (1000 U/mL) or vehicle control and IFITM3 measured (representative immunoblot shown from n = 4 independent experiments). (G and H) sh-NC or sh-IFITM3 MEG-01s were stimulated with PMA and IFN-α and incubated with fibrinogen. Fibrinogen endocytosis (percentage change in cells with endocytosed fibrinogen and MFI compared with unstimulated sh-NC or sh-IFITM3 MEG-01) was measured in mature CD41+ MEG-01s by flow cytometry (n = 5–6). Paired t test. (I) CD34+-derived megakaryocytes (Mks) were treated with vehicle or IFN-α (1000 U/mL) for 24 hours before incubation with labeled dextran. Dextran endocytosis (%) was measured in CD41+ megakaryocytes by flow cytometry (n = 6). Mann-Whitney U test. (J) PMA-differentiated MEG-01s were stimulated with IFN-α (1000 U/mL) and 24 hours later incubated with labeled dextran. CD41+ MEG-01s were measured for dextran endocytosis by flow cytometry (n = 4 per group). Unpaired t test. *P ≤ 0.05; **P ≤ 0.01